U.S. patent number 6,638,758 [Application Number 09/876,199] was granted by the patent office on 2003-10-28 for process for the enzymatic resolution of lactams.
This patent grant is currently assigned to G.D. Searle & Co.. Invention is credited to Alok K. Awasthi, Rolando E. Gapud, Donald W. Hansen, Jr., John S. Ng, Mahima Trivedi, Ping T. Wang.
United States Patent |
6,638,758 |
Hansen, Jr. , et
al. |
October 28, 2003 |
Process for the enzymatic resolution of lactams
Abstract
A method of separating enantiomeric lactam esters. The lactam
esters are contacted with a biocatalyst, such as an enzyme or a
microorganism, in a solution wherein only one enantiomer is
selectively hydrolyzed to give the optically active isomer of the
corresponding acid. The hydrolysis product is then separated from
the unreacted lactam esters. The enzyme is then recycled for reuse
in the next enzymatic resolution. The undesired isomer is also
racemized and reused in the next enzymatic resolution.
Inventors: |
Hansen, Jr.; Donald W. (Skokie,
IL), Trivedi; Mahima (Glenview, IL), Gapud; Rolando
E. (Chicago, IL), Ng; John S. (Chicago, IL), Awasthi;
Alok K. (Skokie, IL), Wang; Ping T. (Manchester,
MO) |
Assignee: |
G.D. Searle & Co. (Skokie,
IL)
|
Family
ID: |
22121926 |
Appl.
No.: |
09/876,199 |
Filed: |
June 6, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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250197 |
Feb 16, 1999 |
6277626 |
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Current U.S.
Class: |
435/280; 435/117;
435/120; 435/121 |
Current CPC
Class: |
C12P
17/10 (20130101); C12P 17/12 (20130101); C12P
41/005 (20130101) |
Current International
Class: |
C12P
41/00 (20060101); C12P 17/10 (20060101); C07C
001/04 (); C12P 017/00 () |
Field of
Search: |
;435/280,117,120,121 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0115860 |
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Feb 1984 |
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EP |
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0634492 |
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Sep 1994 |
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EP |
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57198098 |
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Dec 1982 |
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JP |
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06 199221 |
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Dec 1992 |
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JP |
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11 113594 |
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Oct 1997 |
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JP |
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WO9400362 |
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Jan 1991 |
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WO |
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WO9403428 |
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Feb 1994 |
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WO |
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WO9829561 |
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Jul 1998 |
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WO |
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Other References
C Roberge, et al., Chemical Abstracts, vol. 126 (17): 23g, 1997.
.
W. Boland, et al., Synthesis, 1049-1072, 1991. .
H. Ohta, et al., Chemistry Letters, 657-660, 1992. .
M. Shimoda, et al., Tetrahendron Letters, vol. 29 (52):6961-6964,
1988. .
H. Hemmerle & H. Gais, Tetrahendron Letters, vol. 28 (30):
3471-3474, 1987. .
B. Brion, et al., Tetrahendron Letters, vol. 33 (34): 4889-4892,
1992. .
A.J. Pearson, et al., JOC, 54:3882, 1989. .
C. Sih, et al., JOC, 58: 1068, 1993. .
Bryan et al., Proteins: Structure, Function, Genetics, 1 (4):
326-334, 1986. .
K. Babiak et al., Journal of Organic Chemistry, vol. 55: 3357-81,
1990. .
Basak, et al., Tetrahendron Letters, vol. 38 (4): 643-646, 1997.
.
Dugas, Canadian Journal of Biochem., 47: 985-987, 1969..
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Primary Examiner: Marx; Irene
Attorney, Agent or Firm: Polster, II; Philip B.
Parent Case Text
This is a continuation of application Ser. No. 09/250,197 filed
Feb. 16, 1999 now U.S. Pat No. 6,277,626; which claims priority to
U.S. Provisional Application 60/074,830 filed Feb. 17, 1998.
Claims
We claim:
1. A method of separating enantiomeric lactam esters, wherein the
lactams are of the formula (V): ##STR7## R.sup.1 is selected from
the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, which may optionally be substituted by one or more of
the following: alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol,
thioalkoxy, and halogen; and R.sup.2, R.sup.3, and R.sup.4 are
independently selected from alkyl, alkenyl, alkynyl, hydroxy,
alkoxy, thiol, thioalkoxy, halogen, nitro, amino, alkylamino,
dialkylamino, aminoalkyl, dialkylaminoalkyl, cyano, and haloalkyl,
wherein all said substitutions may be optionally substituted with
one or more of the following: halogen, alkyl, amino, alkylamino,
dialkylamino, aminoalkyl, hydroxy, and alkoxy, comprising
contacting the lactam esters with a biocatalyst in an aqueous
solution, an organic solvent, or a mixture of organic and aqueous
solvents wherein only one enantiomer is selectively hydrolyzed to
give the optically active isomer of the corresponding acid, and
separating the hydrolysis product from the unreacted lactam esters.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the enzymatic resolution of
lactams. The method of the present invention is useful in preparing
compounds which may have utility as pharmaceutical, agricultural
and veterinary products or starting materials and intermediates for
their synthesis.
2. Discussion of the Prior Art
It is known in the art that chiral resolution of compounds can be
achieved by using enzymes. Chiral resolution using enzymes such as
esterases on aliphatic esters and cyclic compounds containing
esters are described in, for example, W. Boland et al., Synthesis,
1049-1072, 1991. Chiral resolutions using enzymes in aliphatic
methyl esters hydrolysis is described in, for example, H. Ohta et
al., Chem. Lett.,657-660, 1992. Chiral resolutions using enzymes in
cyclohexanes systems are described in, inter alia, M. Ohno, Tet.
Lett., 29, 6961-6964, 1988; H. Hemmerle, Tet Lett., 28, 3471-3474,
1987; and B. Brion, Tet Lett., 33, 4889-4892, 1992. Chiral
resolutions using lipases or Acetylcholine esterases on
cycloheptanes containing diacetates is found in A. J. Pearson et
al., JOC, 54, 3882, 1989. Beta-lactams have been reported to be
selectively acylated by lipase at the nitrogen function (C. Sih et
al., JOC, 58, 1068, 1993).
However, there is no prior art for the enzymatic resolution of
lactam esters. It is often desired to obtain a single enantiomer of
a racemic lactam ester. These compounds can be used as
intermediates for preparing compounds which have utility as
starting materials and intermediates for the synthesis of
pharmaceutical, agricultural and veterinary products. For example,
the enanantiomerically pure form of 7-carbomethoxycaprolactam is a
useful intermediate in the synthesis of pharmaceutical drug
candidates.
SUMMARY OF THE INVENTION
The present invention is directed to a method of separating
enantiomeric lactam esters. The lactam esters are contacted with a
biocatalyst, such as an enzyme or a microorganism, in an aqueous
solution, an organic solvent, or a mixture of organic and aqueous
solvents, wherein only one enantiomer is selectively hydrolyzed to
give the optically active isomer of the corresponding acid. The
hydrolysis product is then separated from the unreacted lactam
esters using standard methods known to those skilled in the art.
This invention also discloses a novel method for the recycling and
re-use of the enzymes as well as the racemization of either
enantiomer of the lactam ester after enzymatic resolution.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is directed to contacting racemic esters of
lactam with a biocatalyst, such as an enzyme or a microorganism,
whereby one of the optical isomers is selectively hydrolyzed to
give the optically active isomer of the corresponding acid. The
optically active products are then isolated/purified using suitable
procedures.
For illustrative purposes only, the process of the present
invention is demonstrated by the following example of enzymatic
cleavage of a racemic 7-carbomethoxy caprolactam wherein the S
configuration is converted to its acid: ##STR1##
For convenience, the procedure is described herein using racemic
esters of lactam; however, the method of the present invention is
not limited to use with the racemic form. The lactam ester may be
present in the optical active form or in nonracemic mixtures which
have an excess of one of the optical isomers. The method of the
present invention allows the lactam mixture to react such that only
one of two enantiomeric esters of the lactam is converted to its
acid.
The term "stereoselective hydrolysis" refers to the preferential
hydrolysis of one enantiomer relative to another. The term
"mixture" as used herein in relation to enantiomeric compounds,
denotes mixtures having equal (racemic) or nonequal amounts of
enantiomers. The term "resolution" denotes partial, as well as,
preferably, complete resolution. The term "enzymatic process" or
"enzymatic method" or "enzymatic reaction" denote a process or
method or reaction of the present invention employing an enzyme or
microorganism. The term "enantiomeric excess(es)" is related to the
older term "optical purity". In a mixture of a pure enantiomer (R
or S) and a racemate, enantiomeric excess is the percent excess of
the enantiomer over the racemate. It can be expressed in the
following equation, for example: ##EQU1##
The enzyme may be any enzyme obtainable from animals, plants,
microorganisms, etc. The enzyme may be employed in any conventional
form such as in a purified form, a crude form, a mixture with other
enzymes, a microbial fermentation broth, a fermentation broth, a
microbial body, a filtrate of fermentation broth, and the like,
either solely or in combination. In addition, the enzyme or
microbial body may be immobilized on a resin.
The activities of the enzymes used in this invention are expressed
in "units". Units are defined as the rate of hydrolysis of
p-nitrophenyl proprionate per minutes as expressed in .mu.mol/min
at room temperature.
Specific examples of the enzyme are those obtained from animal and
plants such as cow liver esterase, pig liver esterase, pig pancreas
esterase, horse liver esterase, dog liver esterase, pig
phosphatase, amylase obtainable from barley and potato and lipase
obtainable from wheat. Other examples are hydrolases obtained from
such microorganisms as Rhodotorula, Trichoderma, Candida,
Hansenula, Pseudomonas, Bacillus, Achromobacter, Nocardia,
Chromobacterium, Flavobacterium, Rhizopus, Mucor, Aspergillus,
Alkaligenes, Pediococcus, Klebsiella, Geotrichum, Lactobaccilus,
Cryptococcus, Pichia, Aureobasidium, Actinomucor, Enterobacter,
Torulopsis, Corynebacterium, Endomyces, Saccaromyces, Arthrobacter,
Metshnikowla, Pleurotus, Streptomyces, Proteus, Gliocladium,
Acetobacter, Helminthosporium, Brevibacterium, Escherichia,
Citrobacter, Absidia, Micrococcus, Microbacterium, Penicillium and
Schizophyllium as well as from lichen and algae.
Specific examples of the microorganisms useful in the present
invention include, but are not limited to, Rhodotorula minuta,
Rhodotorula rubra, Candida krusei, Candida cylindracea, Candida
tropicalis, Candida utilus, Pseudomonas fragi, Pseudomonas putida,
Pseudomonas fluorescens, Pseudomonas aeruginosa, Rhizopus
chinensis, Mucor pusillus, Aspergillus niger, Alkaligenes faecalis,
Torulopsis ernobii, Bacillus cereus, Bacillus subtilis, Bacillus
pulmilus, Bacillus subtilis var. niger, Citrobacter freundii,
Micrococcus varians, Micrococcus luteus, Pediococcus acidlactici,
Klebsiella pneumoriae, Absidia hyalospora, Geotrichun candidum,
Schizophyllum commune, Nocardia uniformis subtsuyanarenus, Nocardia
uniformis, Chromobacterium chocolatum, Hansenula anomala var.
ciferrii, Hansenula anomala, Hansenula polymorpha, Achromobacter
lyticus, Achromobacter parvulus, Achromobacter sinplex, Torulopsis
candida, Corynebacterium sepedonicum, Endomyces geotrichum,
Saccaromyces carrvisial, Arthrobacter globiformis, Streptomyces
grisens, Micrococcus luteus, Enterobacter cloacae, Corynebacterium
ezui, Lacto bacillus casei, Cryptococcus albidus, Pichia
polimorpha, Penicillium frezuentans, Aureobasidium pullulans,
Actinomucor elegans, Streptomyces grisens, Proteus vulgaris,
Gliocladium roseum, Gliocladium virens, Acetobacter aurantius,
Helminthosporium sp. Chromobacterium iodinum, Chromobacterium
violaceum, Flavobacterium lutescens, Metschnikowia pulcherrima,
Pleurotus ostreatus, Brevibacterium ammoniagenes, Brevibacterium
divaricatum, Escherichia coli, Rodotolura minuta var. texensis,
Trichoderma longibrachiatum, Mucor javanicus, Flavobacterium
arbonescens, Flavobacterium heparinum, and Flavobacterium
capsulatum.
Exemplary, commercially available enzymes suitable for use in the
present invention include lipases such as Amano PS-30 (Pseudomonas
cepacla), Amano GC-20 (Geotrichum candidum), Amano APF (Aspergillus
niger), Amano AK (Pseudomonas sp.), Pseudomonas fluorescens lipase
(Biocatalyst Ltd.), Amano Lipase P30 (Pseudomonas sp.), Amano P
(Pseudomonas fluorescens), Amano AY-30 (Candida cylindracea), Amano
N (Rhizopus niveus), Amano R (Penicillium sp.), Amano FAP (Rhizopus
oryzae), Amano AP-12 (Aspergillus nlger), Amano MAP (Mucor melhei),
Amano GC-4 (Geotrichum candidum), Sigma L-0382 and L-3126 (porcine
pancrease), Lipase OF (Sepracor), Esterase 30,000 (Gist-Brocarde),
KID Lipase (Gist-Brocarde), Lipase R (Rhizopus sp., Amano), Sigma
L-3001 (Wheat germ), Sigma L-1754 (Candida cytindracea), Sigma
L-0763 (Chromobacterlum vlscosum) and Amano K-30 (Aspergillus
nlger). Additionally, exemplary enzymes derived from animal tissue
include esterase from pig liver, chymotrypsin and pancreatin from
pancreas such as Porcine Pancreatic Lipase (Sigma). Two or more, as
well as a single, enzyme may be employed when carrying out the
process of the present invention.
In addition, enzymes which are serine carboxypeptidases can be
used. These enzymes are derived from Candida lipolytica,
Saccharomyces cerevisiae, wheat (Triticum aestivum) and Penicillium
janthinellum. Commercially available cross-linked enzyme crystals
may also be used such as from Altus Biologics, Inc.(e.g.
ChiroCLEC-CR, ChiroCLEC-PC, ChiroCLEC-EC).
The present invention is also directed to the use of thermostable
esterases and genetically engineered esterases for the resolution
of the lactam esters. These enzymes, commercially available from
ThermoGen, Inc., are especially suitable for use in industrial
processes and are easy to use. In addition to functioning at a wide
range of temperatures including higher temperatures, these
thermostable enzymes possess an increased shelf life which improves
handling. The enzymes are also able to endure harsh, non-biological
conditions (pH, salt concentrations, etc.) usually associated with
industrial processes because of their stability under operational
conditions. They can be immobilized for reuse in multiple
applications and hence improving the cost-effectiveness of the
process.
During the isolation of the products after enzymatic resolution,
the enzymes are frequently exposed to traces of organic solvents.
In addition, some enzymatic resolutions are found to work best
under a mixture of aqueous and organic solvents or in organic
solvents alone. The esterase enzymes of the present invention are
more tolerant to denaturing by many organic solvents compared to
conventional enzymes which allows longer operational half lives.
Most of the esterase enzymes are produced using genetic engineering
techniques of gene cloning which ensures the purity of these
enzymes and the ease of process controls during scale up.
It was discovered that many esterase and lipase enzymes offer a
high degree of stereoselectivity in the resolution of the lactam
esters. The preferred enzymes for the resolutions of lactam esters
include the thermoesterases THERMOCAT E002, THERMOCAT E010,
THERMOCAT E015, THERMOCAT E020 from ThermoGen, Inc. with the most
preferred enzymes being the THERMOCAT E020.
Instead of isolated enzymes, there may also be employed a
microorganism which can produce any enzyme as stated above.
The enzyme or microorganisms may be used alone or in combination.
Depending upon the type of enzyme or microorganism used, either one
of the optical isomers of the lactam ester is predominantly
hydrolyzed to give the optically active acid. Either one of the
optical isomers may be obtained by the selection of a suitable
enzyme or microorganism.
The enzymatic hydrolysis of the present invention may be carried
out by contacting the lactam esters with the enzyme or
microorganism, usually in an aqueous buffer medium with good
agitation.
The buffer medium may be inorganic acid salt buffers (e.g.
potassium dihydrogen phosphate, sodium dihydrogen phosphate),
organic acid salt buffers (e.g. sodium citrate), or any other
suitable buffer. The concentration of the buffer may vary from
0.005 to 2 M, preferably from 0.005 to 0.5 M and will depend on the
specific lactam ester and the enzymes microorganism used.
Depending on the solubility of the lactam esters, a surfactant may
be added to the reaction mixture to solubilize the substrate;
preferred surfactants include but are not limited to nonionic
surfactants such as alkylaryl polyether alcohols. A preferred
surfactant is octylphenoxy polyethoxyethanol, commercially
available as Triton X-100 (from Sigma Chemical Company). An
effective amount of a surfactant is used. Typical amounts can vary
from 0.05% to about 10%.
It is sometimes preferable to add an effective amount of an organic
cosolvent to increase product solubility to facilitate the
reaction. Examples of solvents include but are not limited to
acetonitrile, THF, DMSO, DMF, alcohols, etc. Effective amounts of a
co-solvent includes from 1% to 30% depending on the specific lactam
ester and enzymes and/or microorganism used.
The pH of the buffers or the pH of the reaction is normally from 4
to 10, preferably from 5 to 9, most preferably from 7 to 8. The
reaction temperature may vary from 0 to 100.degree. C. and will
depend on the specific lactam ester and the enzymes or
microorganism used. The reaction time is generally from 1 hour to
70 hours and will depend on the specific lactam ester, enzyme
concentration and the enzymes the microorganism used. Normally, the
enzymatic hydrolysis is allowed to proceed for a period sufficient
to generate a satisfactory quantity of the desired esters or acid
in satisfactory optical purity. As the reaction progresses, the
amount of desired ester or acid and their optical purities may be
monitored by HPLC and chiral HPLC. Normally, the conversion is
carried to approximately 50%, after which the acid and the esters
are usually obtained in good yields after isolation.
The amount of enzyme used could vary widely from 5 units to 12,000
units of enzyme per mole of starting materials. (The activities of
the enzymes used in this invention are expressed in "units". Units
are defined as the rate of hydrolysis of p-nitrophenyl proprionate
per minutes as expressed in .mu.mol/min at room temperature). The
amount of enzyme needed will depend on the temperature, the
specific lactam ester, the enzymes and/or microorganism used, and
the desirable reaction time. It may also be desirable to use a
large amount of enzymes in some cases to ensure a practically short
reaction time, especially when the enzymes are immobilized and can
be reused for many turnovers. The concentration of the ester
substrate may be from 0.1 g/L to 100 g/L and depends on the
specific lactam ester and the enzyme and/or microorganism used.
The enzymes and/or microorganisms used in the present invention may
be in crude form or in an immobilized form. They can be immobilized
on various solid supports without loss of stereospecificity or
change in stereo selectivity. The solid supports can be inert
absorbents to which the enzyme is not covalently bonded. Instead
the enzyme is absorbed such as by interactions of hydrophobic or
hydrophilic portions of a protein with like regions of the inert
absorbent, by hydrogen bonding, by salt bridge formation, or by
electrostatic interactions. Inert absorbent materials include, but
are not limited to, synthetic polymers (e.g. polystyrene,
poly-(vinylalcohol), polyethylene and polyamides), mineralaceous
compounds (e.g. diatomaceous earth and Fuller's earth), or
naturally occurring polymers (e.g. cellulose). Specific examples of
such materials include Celite 545 diatomaceous earth, Abelite XAD-8
polymeric resin beads and polyethylene glycol 8000.
The enzyme may also be immobilized on the support to which the
enzyme is covalently bonded (e.g., oxirane-acrylic beads and
glutaraldehyde activated supports). Specific examples include
Eupergit C oxirane-acrylic beads and glutaraldehyde activated
Celite 545. Other possible immobilizing systems are well known and
are readily available to those skilled in the art of enzyme
immobilization.
Instead of conventional immobilization method described above, it
was discovered that the enzymes could also be conveniently recycled
for reuse by simply precipitating out the used enzymes with
ammonium sulfate. The precipitated enzyme-ammonium sulfate could be
used directly in the next enzymatic hydrolysis. Salts are commonly
used in purification of enzymes. They generally protect the protein
enzymes by reducing solvent activity. It was discovered that
ammonium sulfate, potassium sulfate, potassium phosphate, sodium
chloride etc. are effective in recovering the enzyme thermoesterase
(E020) activity. Among the salts, ammonium sulfate is the most
preferred one.
The desired products, the optically pure (or enriched) unreacted
ester and the optically pure (or enriched) acid may be isolated
from the hydrolysis mixture using conventional methods such as
extractions, acid-base extractions, filtration, chromatography,
crystallization or combinations thereof. The recovered enzyme or
microorganism may be recycled as described above.
In a convenient isolation procedure, after the enzymatic
hydrolysis, the pH is adjusted to pH 7.5 to 8 (in the case of
immobilized biocatalysts, the biocatalyst is first separated by
filtration), the product acid is separated from the unreacted ester
by extracting the ester with an organic solvent such as methylene
chloride, ethyl acetate, diethyl ether, methyl t-butyl ether, or
any other solvent in which the substrate is soluble and stable.
Concentration of the organic extracts affords the optically pure
(or enriched) unreacted ester. Concentration of the aqueous phase
yield the optically pure (or enriched) acid.
The acid can be freed of the buffer salts and enzyme by selective
precipitation or chromatography or other methods known to those
skilled in the art. These include acidifying the aqueous to pH 3
(or lower) and isolating the acid by extracting the acid with
organic solvent such as methylene chloride, ethyl acetate, diethyl
ether, methyl t-butyl ether, or any other solvent in which the acid
is soluble and stable. Concentration of the organic extracts
affords the optically pure (or enriched) unreacted ester and the
optically pure (or enriched) acid and which can be purified and
freed of the buffer salts and enzyme by selective precipitation or
chromatography or other methods known to those skilled in the
art.
Either of the optically pure (or enriched) unreacted ester and the
optically pure (or enriched) acid could be racemized if so desired.
The optically pure (or enriched) unreacted ester could be racemized
by heating in the appropriate base under appropriate conditions.
Alternatively, the optically pure (or enriched) unreacted ester
could be racemized by heating in acid in the presence of an alcohol
under appropriate conditions. The optically pure (or enriched)
unreacted acid could also be racemized and converted to the racemic
esters by heating in acid in the presence of an alcohol under
appropriate conditions. In this manner, excellent yields can be
achieved of either the optically pure (or enriched) unreacted ester
or the optically pure (or enriched) acid by this combination of
stereoselective enzymatic hydrolysis and racemization
techniques.
In a preferred embodiment, the present invention is directed to the
enzymatic ##STR2## R.sup.1 is selected from the group consisting of
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl,
aryl, and heteroaryl which may optionally be substituted by one or
more of the following alkyl, alkenyl, alkynyl, hydroxy, alkoxy,
thiol, thioalkoxy, halogen, nitro, amino, alkylamino, dialkylamino,
aminoalkyl, dialkylaminoalkyl, arylamino, aminoaryl,
alkylaminoaryl, cyano, haloalkyl; A is selected from the group
consisting of O, S, and NH or N which may be substituted
respectively with one or two independent R.sup.1 (the R.sup.1 s
need not be the same); L is selected from the group consisting of
no group or alkylene, alkenylene, alkynylene, and
--(CH.sub.2).sub.m --D--(CH.sub.2).sub.n --; D is selected from the
group consisting of O, S, SO, SO.sub.2, Se, SeO, SeO.sub.2,
N--R.sup.6 ; R.sup.6 is selected from the group consisting of
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
heterocyclyl, aryl, and heteroaryl which may optionally be
substituted by one or more of the following: alkyl, alkenyl,
alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, halogen, nitro, amino,
alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, arylamino,
aminoaryl, alkylaminoaryl, cyano, haloalkyl; m=0 to about 7; n=1 to
about 5; wherein L may optionally be substituted by one or more of
the following: alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol,
thioalkoxy, S(O)R.sup.7, S(O).sub.2 R.sup.7, halogen, nitro, amino,
alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, arylamino,
aminoaryl, alkylaminoaryl, cyano, haloalkyl, wherein all said
substitutions may be optionally substituted with one or more of the
following: alkyl, amino, alkylamino, dialkylamino, aminoalkyl, and
R.sup.7 is alkyl, or aryl; X is selected from the group consisting
of NH, O, S, Se, (CH.sub.2).sub.p, and CH.dbd.CH; p=0 to about 4; Y
is selected from the group consisting of NH, O, S, SO, SO.sub.2,
Se, SeO, SeO.sub.2, (CH2).sub.q, CH.dbd.CH; q=0 to about 2; Z is
selected from the group consisting of (CH.sub.2).sub.v, CH.dbd.CH;
v=0 to about 2; R.sup.2, R.sup.3, and R.sup.4 are independently
selected from alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol,
thioalkoxy, S(O)R.sup.7, S(O).sub.2 R.sup.7, halogen, nitro, amino,
alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, arylamino,
aminoaryl, alkylaminoaryl, cyano, haloalkyl, wherein all said
substitutions may be optionally substituted with one or more of the
following: halogen, alkyl, amino, alkylamino, dialkylamino,
aminoalkyl, hydroxy, alkoxy, and R.sup.5 is independently selected
from hydrogen, alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol,
thioalkoxy, S(O)R.sup.7, S(O).sub.2 R.sup.7, halogen, nitro, amino,
alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, arylamino,
aminoaryl, alkylaminoaryl, cyano, haloalkyl, wherein all said
substitutions may be optionally substituted with one or more of the
following: halogen, alkyl, amino, alkylamino, dialkylamino,
aminoalkyl, hydroxy, alkoxy; R.sup.2, R.sup.3, may optionally be
taken together to form an alicyclic hydrocarbon, heterocyclyl,
heteroaryl or aromatic hydrocarbon and said optionally formed ring
may be optionally substituted with one or more of alkyl, alkenyl,
alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, S(O)R.sup.7,
S(O).sub.2 R.sup.7, halogen, nitro, amino, alkylamino,
dialkylamino, aminoalkyl, dialkylaminoalkyl, arylamino, aminoaryl,
alkylaminoaryl, cyano, or haloalkyl.
Preferably, the present invention is directed to the enzymatic
resolution of lactams ##STR3## R.sup.1 is selected from the group
consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
which may optionally be substituted by one or more of the
following: alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol,
thioalkoxy, and halogen; L is selected from the group consisting of
no group or alkylene, alkenylene, and alkynylene; wherein L may
optionally be substituted by one or more of the following: alkyl,
alkenyl, alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, halogen,
nitro, amino, alkylamino, dialkylamino, aminoalkyl,
dialkylaminoalkyl, arylamino, aminoaryl, alkylaminoaryl, cyano, and
haloalkyl; X is selected from the group consisting of
(CH.sub.2).sub.p, and CH.dbd.CH; p=0 to about 4; Y is selected from
the group consisting of NH, O, (CH.sub.2).sub.q and CH.dbd.CH; q=0
to about 2; Z is selected from the group consisting of
(CH.sub.2).sub.v and CH.dbd.CH; v=0 to about 2; R.sup.2, R.sup.3,
and R.sup.4 are independently selected from alkyl, alkenyl,
alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, halogen, nitro, amino,
alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, cyano, and
haloalkyl, wherein all said substitutions may be optionally
substituted with one or more of the following: halogen, alkyl,
amino, alkylamino, dialkylamino, aminoalkyl, hydroxy, and
alkoxy;
More preferably the present invention is directed to the enzymatic
resolution of ##STR4## R.sup.1 is selected from the group
consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
which may optionally be substituted by one or more of the
following: alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol,
thioalkoxy, and halogen; Y is selected from the group consisting of
NH, O, (CH.sub.2).sub.q and CH.dbd.CH; q=0 to about 2; Z is
selected from the group consisting of (CH.sub.2).sub.v and
CH.dbd.CH; v=0 to about 2; R.sup.2, R.sup.3, and R.sup.4 are
independently selected from alkyl, alkenyl, alkynyl, hydroxy,
alkoxy, thiol, thioalkoxy, halogen, nitro, amino, alkylamino,
dialkylamino, aminoalkyl, dialkylaminoalkyl, cyano, and haloalkyl,
wherein all said substitutions may be optionally substituted with
one or more of the following: halogen, alkyl, amino, alkylamino,
dialkylamino, aminoalkyl, hydroxy, and alkoxy.
Even more preferably, the present invention is directed to the
enzymatic resolution of lactams of the formula (IV): ##STR5##
R.sup.1 is selected from the group consisting of alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, which may optionally be
substituted by one or more of the following: alkyl, alkenyl,
alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, and halogen; Y is
selected from the group consisting of NH, O, (CH.sub.2).sub.q and
CH.dbd.CH; q=0 to about 2; R.sub.2, R.sub.3, and R.sub.4 are
independently selected from alkyl, alkenyl, alkynyl, hydroxy,
alkoxy, thiol, thioalkoxy, halogen, nitro, amino, alkylamino,
dialkylamino, aminoalkyl, dialkylaminoalkyl, cyano, and haloalkyl,
wherein all said substitutions may be optionally substituted with
one or more of the following: halogen, alkyl, amino, alkylamino,
dialkylamino, aminoalkyl, hydroxy, and alkoxy.
Most preferred the present invention is directed to the enzymatic
resolution of lactams of the formula (V): ##STR6## R.sup.1 is
selected from the group consisting of alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, which may optionally be substituted by
one or more of the following: alkyl, alkenyl, alkynyl, hydroxy,
alkoxy, thiol, thioalkoxy, and halogen; R.sup.2, R.sup.3, and
R.sup.4 are independently selected from alkyl, alkenyl, alkynyl,
hydroxy, alkoxy, thiol, thioalkoxy, halogen, nitro, amino,
alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, cyano, and
haloalkyl, wherein all said substitutions may be optionally
substituted with one or more of the following: halogen, alkyl,
amino, alkylamino, dialkylamino, aminoalkyl, hydroxy, and
alkoxy.
As utilized herein, the term "alkyl", alone or in combination,
means an acyclic alkyl radical containing from 1 to 10, preferably
from 1 to 8 carbon atoms and more preferably 1 to 6 carbon atoms.
Examples of such radicals include methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl,
iso-amyl, hexyl, octyl and the like.
The term "alkenyl" refers to an unsaturated acyclic hydrocarbon
radical in so much as it contains at least one double bond. Such
radicals containing from 2 to 10 carbon atoms, preferably from 2 to
8 carbon atoms and more preferably 2 to 6 carbon atoms. Examples of
suitable alkenyl radicals include propylenyl, buten-1-yl,
isobutenyl, pentenylen-1-yl, 2-2-methylbuten-1-yl,
3-methylbuten-1-yl, hexen-1-yl, hepten-1-yl, and octen-1-yl, and
the like.
The term "alkynyl" refers to an unsaturated acyclic hydrocarbon
radical in so much as it contains one or more triple bonds, such
radicals containing 2 to 10 carbon atoms, preferably having from 2
to 8 carbon atoms and more preferably having 2 to 6 carbon atoms.
Examples of suitable alkynyl radicals include ethynyl, propynyl,
butyn-1-yl, butyn-2-yl, pentyn-1-yl, pentyn-2-yl,
3-methylbutyn-1-yl, hexyn-1-yl, hexyn-2-yl, hexyn-3-yl,
3,3-dimethylbutyn-1-yl radicals and the like.
The term "heterocyclyl" means an unsaturated cyclic hydrocarbon
radical with 3 to about 6 carbon atoms, wherein 1 to about 4 carbon
atoms are replaced by nitrogen, oxygen or sulfur. The
"heterocyclyl" may be fused to an aromatic hydrocarbon radical.
Suitable examples include pyrrolyl, pyridinyl, pyrazolyl,
triazolyl, pyrimidinyl, pyridazinyl, oxazolyl, thiazolyl,
imidazolyl, indolyl, thiophenyl, furanyl, tetrazolyl, 2-pyrrolinyl,
3-pyrrolinyl, pyrrolindinyl, 1,3-dioxolanyl, 2-imidazolinyl,
imidazolidinyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl,
isothiazolyl, 1,2,3-oxadiazolyl, 1,2,3-triazolyl,
1,3,4-thiadiazolyl, 2H-pyranyl, 4H-pyranyl, piperidinyl,
1,4-dioxanyl, morpholinyl, 1,4-dithianyl, thiomorpholinyl,
pyrazinyl, piperazinyl, 1,3,5-triazinyl, 1,3,5-trithianyl,
benzo(b)thiophenyl, benzimidazonyl, quinolinyl, and the like.
The term "aryl" means an aromatic hydrocarbon radical of 4 to 16
carbon atoms, preferably 6 to about 12 carbon atoms, more
preferably 6 to 10 carbon atoms. Examples of suitable aromatic
hydrocarbon radicals include phenyl, naphthyl, and the like.
The term "heteroaryl" means aromatic hydrocarbon radical of 4 to 16
carbon atoms, preferably 6 to about 12 carbon atoms, more
preferably 6 to 10 carbon atoms wherein 1 to about 4 carbon atoms
are replaced by nitrogen, oxygen or sulfur.
The terms "cycloalkyl" or "cycloalkenyl" means an "alicyclic
radical in a ring with 3 to 10 carbon atoms, and preferably from 3
to 6 carbon atoms. Examples of suitable alicyclic radicals include
cyclopropyl, cyclopropylenyl, cyclobutyl, cyclopentyl, cyclohexyl,
2-cyclohexen-1-ylenyl, cyclohexenyl and the like.
The term "alkoxy", alone or in combination, means an alkyl ether
radical wherein the term alkyl is as defined above and most
preferably containing 1 to 4 carbon atoms. Examples of suitable
alkyl ether radicals include methoxy, ethoxy, n-propoxy,
isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy and the
like.
The term "halogen" means fluorine, chlorine, bromine or iodine.
The term "prodrug" refers to a compound that is made more active in
vivo.
As used herein, reference to "treatment" of a patient is intended
to include prophylaxis.
EXAMPLES
Example 1
Enzymatic Resolution of Racemic 7-carbomethoxycaprolactam
Four enzymes were each dissolved in 20 mL of buffer (pH 7, Sigma
phosphate buffer with conc. of 0.1 mol/liter) to form four separate
solutions. Racemic 7-carbomethoxycaprolactam was added to each
solution. Each solution was then allowed to react at room
temperature (20-25.degree. C.). Aliquots were drawn (non-uniform in
volume) at specific time intervals and their pH measured.
Each aliquot was then acidified to pH=1 with 0.5N KHSO.sub.4 and
then extracted with methylene chloride. The organic layer was
separated, dried (MgSO.sub.4), filtered, stripped in vacuo and
submitted for HPLC analysis. The mass recovery averaged 60%. The
results are shown in Table 1.
TABLE 1 Chiral HPLC Assay By Area Percent at Varying Time Intervals
HPLC HPLC HPLC HPLC Area Area Area Area Rxn Time % Acid A % Acid B
% Ester A % Ester B pH Altus Biologics Enzyme #13 (Candida
Antarctica B. Lipase) 25 mg of Enzyme per 500 mg Caprolactam
methylester T = 15 min 18.3 6.6 25.8 49.2 7 T = 30 min 26.2 12.0
15.8 45.9 5 T = 1 H 29.2 16.4 12.2 42.1 4 T = 2 H 31.2 21.0 8.7
38.7 3 T = 4 H 31.5 21.3 6.9 40.2 3 Altus Biologics Enzyme #13
(Candida Antarctica B. Lipase) 50 mg of Enzyme per 500 mg
Caprolactam methylester T = 15 min 26.6 11.9 18.2 43.0 5 T = 30 min
29.9 15.5 14.6 39.7 4 T = 1 H 29.4 16.7 12.8 40.7 3-4 T = 2 H 33.7
20.9 8.6 36.7 3 T = 4 H 35.3 23.1 0.0 41.7 3 Altus Biologics Enzyme
#16 (Chiroclec-BL) 4 mg of Enzyme per 500 mg Caprolactam
methylester T = 15 min 11.7 6.7 35.1 46.4 7-8 T = 30 min 19.7 12.1
26.8 41.1 7-8 T = 1 H 20.7 13.4 25.3 40.6 6 T = 2 H 21.7 14.8 23.5
40.1 5 T = 4 H 22.6 15.8 22.5 39.2 4-5 Altus Biologics Enzyme #16
(Chiroclec-BL) 8 mg of Enzyme per 500 mg Caprolactam methylester T
= 15 min 16.6 9.5 31.9 42.1 7-8 T = 1 H 24.5 15.7 23.0 36.8 5-6 T =
1.5 H 23.3 16.1 23.0 37.6 5 T = 2 H 23.6 14.6 23.3 38.5 4-5 T = 4 H
22.0 14.0 23.8 40.3 4-5
Example 2
Enzymatic Resolution of Racemic 7-carbomethoxycaprolactam
Seven enzymes were each dissolved in 50 mL of buffer (pH 7)
(phosphoric acid added to Sigma phosphate buffer with conc. of 0.1
mol/liter and pH 7.4) to form seven separate solutions. Racemic
7-carbomethoxycaprolactam was added to each solution so that 500
units of enzyme were used per 500 mg of ester substrate. Each
solution was then allowed to react at room temperature
(20-25.degree. C.). Aliquots were drawn (non-uniform in volume) at
specific time intervals and their pH measured.
Each aliquot was then acidified to pH=1 with 1M KHSO.sub.4 and then
extracted with methylene chloride. The organic layer was separated,
dried (MgSO.sub.4), filtered, stripped in vacuo and submitted for
HPLC analysis. The mass recovery averaged 50-60%. The results are
shown in Table 2.
TABLE 2 Extraction Work Up THERMOGEN HYDROLYSIS ENZYMES (500 U WITH
500 mg Caprolactam methylester) HPLC HPLC HPLC HPLC Area Area Area
Area Enzyme # % Acid A % Acid B % Ester A % Ester B T = 2 H E001
0.8 0.0 28.0 38.9 E002 2.2 0.0 37.3 59.1 E006 1.7 0.0 40.1 57.7
E009 0.0 0.0 46.8 51.8 E010 9.9 0.5 30.4 59.3 E014 1.7 0.4 44.6
53.4 E015 6.1 0.3 35.3 57.8 T = 4 H E001 3.5 0.0 20.7 52.5 E002
11.0 0.6 21.0 65.8 E006 12.8 0.7 19.3 66.9 E009 1.0 0.0 44.8 53.3
E010 14.6 1.1 14.2 70.1 E014 3.0 0.2 41.5 55.3 E015 2.7 0.3 8.5
23.8 T = 9 H E001 37.4 4.9 2.6 53.2 E002 35.7 4.6 3.0 55.8 E006
12.2 1.8 0.8 17.4 E009 9.6 1.6 34.9 46.6 E010 23.1 3.0 1.9 72.1
E014 5.1 3.2 36.8 54.9 E015 18.9 5.4 8.9 66.9 T = 20 H E001 33.3
8.4 0.5 57.0 E002 32.5 6.7 0.3 55.6 E006 24.9 4.6 1.2 59.0 E009 6.1
0.7 31.9 58.0 E010 21.9 3.1 0.0 75.0 E014 4.4 0.6 31.4 63.6 E015
14.2 1.3 4.1 80.4
Four of the enzymes were also separately worked up with 1M
KHSO.sub.4 and then lyophilized. The residue was extracted
extensively with methylene chloride. The extracts were stripped in
vacuo and submitted for HPLC analysis. This method improved mass
recovery to about 90%. The results are shown in Table 3. As can be
seen in Table 3, the pH drops steadily during the hydrolysis
reaction.
TABLE 3 Lyophilization Work Up HPLC HPLC HPLC HPLC Area Area Area
Area mg Rxn Time % Acid A % Acid B % Ester A % Ester B covered pH
THERMOGEN HYDROLYSIS ENZYME E002 (500 U with 500 mg Caprolactam
methylester) T = 2 H 21.6 1.8 28.7 47.9 75.0 6.8 T = 4 H 35.2 2.2
14.0 48.7 80.0 6.2 T = 8 H 51.4 4.1 2.5 42.0 305.0 6.2 THERMOGEN
HYDROLYSIS ENZYME E010 (500 U with 500 mg Caprolactam methylester)
HPLC HPLC HPLC Area Area Area mg Rxn Time % Acid A ept at pH %
Ester A % Ester B covered pH T = 2 H 19.9 2.7 30.2 46.1 85.0 6.8 T
= 4 H 33.0 3.1 15.1 47.7 79.0 6.6 T = 8 H 42.3 3.5 3.5 49.6 301.0
6.5 THERMOGEN HYDROLYSIS ENZYME E015 (500 U with 500 mg Caprolactam
methylester) HPLC HPLC HPLC HPLC Area Area Area Area mg Rxn Time %
Acid A % Acid B % Ester A % Ester B covered pH T = 2H 15.8 1.1 36.1
45.9 91.0 6.8 T = 4H 27.8 1.4 20.8 48.7 82.0 6.3 T = 8H 37.8 1.6
11.3 48.7 260.0 6.4 THERMOGEN HYDROLYSIS ENZYME E020 (500 U with
500 mg Caprolactam methylester) HPLC HPLC HPLC HPLC Area Area Area
Area mg Rxn Time % Acid A % Acid B % Ester A % Ester B covered pH T
= 2H 16.0 2.7 32.6 48.5 98.0 6.8 T = 4H 32.3 3.4 13.6 50.7 80.0 6.5
T = 8H 45.4 6.6 1.1 46.4 305.0 6.4
Example 3
Enzymatic Resolution of Racemic 7-carbomethoxycaprolactam
A reaction was conducted using E002 under the conditions set forth
in Example 2 except that a pH stat was employed to maintain a
constant pH of 7 throughout the reaction. The enzyme was worked up
with 1M KHSO.sub.4 and then lyophilized. The residue was extracted
extensively with methylene chloride. The extract was stripped in
vacuo and submitted for HPLC analysis. The results are shown in
Table 4.
TABLE 4 Lyophilization_ Work up THERMOGEN HYDROLYSIS ENZYME E020
(500 U with 500 mg Caprolactam methylester) HPLC HPLC HPLC HPLC
Area Area Area Area Rxn Time % Acid A % Acid B % Ester A % Ester B
T = 2 H 15.8 1.0 33.4 47.0 T = 4 H 28.7 1.7 19.5 50.1 T = 6 H 42.0
3.1 8.2 46.7 T = 8 H 45.3 5.2 3.8 45.0 The reaction was kept at pH
7 using a pH stat. Solvents used were pH 7 phosphate buffer, and
0.1 N NaOH.
Example 4
Enzymatic Resolution of Racemic 7-carbomethoxycaprolactam at
Temperatures Above Room Temperatures
A solution of 33.3 g sodium hydrogenphosphate in 2000 mL
DI(deionized) water was charged to a reaction vessel followed by a
solution of 22.4 g potassium dihydrogenphosphate in 1300 mL DI
water. The reaction mixture was stirred for 15 minutes at 47
C.-48.degree. C. and 2.28 g ThermoCat E020 biocatalyst (activity:
18.5 units/mg) was added. The reaction mixture was stirred for 5 mm
or until a homogeneous solution was obtained. To the above mixture
was added 60 g of 7-carbomethoxy caprolactam and the reaction
mixture was stirred at 47.degree. C.-48.degree. C. The progress of
the reaction was monitored by HPLC. When undesired ester enantiomer
has completely disappeared, which takes 3 to 8 h, the temperature
of reaction mixture was brought down to 25.degree. C.-27.degree. C.
The product mixture was extracted with 3.times.1100 mL
dichloromethane. The organic layer was dried over anhydrous
magnesium sulfate, filtered over celite and concentrated to dryness
below 25.degree. C. to give 22 g of a white waxy solid (73% of
theory). HPLC analysis and comparison with standard samples
indicated the solid isolated was the desired desired
R-enantiomer.
Example 5
Procedures for the Recycling and Re-use of Enzymes
Enzyme Precipitation
A 20-ml reaction mixture containing 2 mmoles of phosphate buffer
(pH 7.6), 140 units of the thermoesterase ThermoCat E020 and 200 mg
of the substrate, 7-carbomethoxy caprolactam, was incubated at
48.degree. C. in a water bath shaker for 2 hours. A control was run
in parallel without the addition of enzyme preparation. At the end
of reaction, a 18 ml aliquot was collected and gently mixed with
7.2 g of ammonium sulfate. After the salt was completely dissolved,
the solution reaches approximately 60% saturation and the protein
enzyme was precipitated. The protein enzyme was then recovered by
centrifugation at 4.degree. C. for 20 minutes.
Recycling and Re-use of Enzyme
The precipitated protein enzyme was re-dissolved in 1.8 mmoles
phosphate buffer (pH 7.6) and tested in a second batch reaction of
an 18 ml re-constituted mixture. The substrate 7-carbomethoxy
caprolactam was reduced to 180 mg and the reaction was followed by
the same procedure as described above. At end of the second
reaction, a 16 ml aliquot was collected and 6.4 g ammonium sulfate
were used to isolate the enzyme. The third recycle reaction was
conducted in a16 ml re-constituted mixture containing 160 mg
substrate by the same procedure. The products are analyzed by HPLC
analyses. The results (HPLC area percentages) of these experimental
are shown in the Table 5:
TABLE 5 HPLC Area HPLC Area % by Species % Total Samples Time acid
ester A ester B acid ester A ester B ee Reaction 0 min. 0 50 50 0
50 50 0% mixture The first reaction: Control 120 min 17 41 41 100
50 50 0% Reaction 120 min 59 8 34 100 18 82 63.6% mixture The
second reaction (first recycle): Control 120 min 40 30 30 100 51 49
0% Reaction 120 min 53 5 42 100 11 89 77.2% mixture The third
reaction (second recycle): Control 120 min 42 32 26 100 56 44 0%
Reaction 120 min 43 15 44 100 25 75 49.3% mixture
From the above data, it can be concluded this enzyme recycling
procedures with ammonium sulfate precipitation is a simple method
to recycle the enzyme for re-use.
Example 6
Procedures for the Racemization of Chiral 7-carbomethoxy
Caprolactam
Method A
To a mixture of 5 microliters of 25% sodium methoxide in methanol
(22 micromoles of sodium methoxide) and 1 mL of dry tetrahydrofuran
(THF) under nitrogen was added 54 mg of optically pure
(R)-7-carbomethoxy caprolactam. The mixture was stirred at room
temperatures for 48 hours. The product was filtered through
Dowex.RTM. 50WX200 ion-exchange resin (Dowex-SOW-hydrogen, strongly
acidic, prewashed with water but not dried), washed with additional
THF and evaporated to dryness to give 44 mg of product. HPLC
analysis of the product indicated that the starting material,
(R)-7-carbomethoxy caprolactam, was completely racemized to a 50/50
mixture of the R and S isomers, (R)-7-carbomethoxy caprolactam and
(S)-7-carbomethoxy caprolactam.
It is understood to those skilled in the art that if one would
start with the (S)-7-carbomethoxy caprolactam as the starting
material, the same racemic product mixture would result.
Method B
To a mixture of 1 mL of dry tetrahydrofuran (THF) and 54 mg of
optically pure (R)-7-carbomethoxy caprolactam under nitrogen was
added 0.47 mL of lithium diisopropylamide(LDA)/THF in cyclohexane
(1.5M, 70 millimoles of LDA). The mixture was stirred at room
temperatures for 1 hour. The product was filtered through
Dowex.RTM. 50WX200 ion-exchange resin (Dowex-50W-hydrogen, strongly
acidic, prewashed with water but not dried), washed with additional
THF and evaporated to dryness to give 34 mg of product. HPLC
analysis of the product indicated that the starting material,
(R)-7-carbomethoxy caprolactam, was completely racemized to a 50/50
mixture of the R and S isomers, (R)-7-carbomethoxy caprolactam and
(S)-7-carbomethoxy caprolactam, along with some racemized
carboxylic acid. It is understood to those skilled in the art that
if one would start with the (S)-7-carbomethoxy caprolactam as the
starting material, the same racemic product mixture would
result.
Method C
To 20 mL of 25% sodium methoxide in methanol was added 64 mg of
optically pure (R)-7-carbomethoxy caprolactam. The mixture was
stirred to reflux for 24 hours. The product was cooled to 0.degree.
C., the product was acidified, extracted with methylene chloride.
The organic layer was concentrated to give 5 mg of solid. The
aqueous layer was concentrated to dryness, the residue solid was
extracted with THF, filtered and concentrated to dryness to give 24
mg of brown oil. HPLC analysis of the product indicated that the
starting material, (R)-7-carbomethoxy caprolactam, has completely
racemized and hydrolzed to a 50/50 mixture of the R and S isomers
of the corresponding racemic acids, (R)-7-carboxy caprolactam and
(S)-7-carboxy caprolactam.
It is understood to those skilled in the art that if one would
start with the (S)-7-carbomethoxy caprolactam as the starting
material, the same racemic product mixture would result.
* * * * *